CN110551653A - method for preparing mycoplasma bovis antigen without serum - Google Patents
method for preparing mycoplasma bovis antigen without serum Download PDFInfo
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- CN110551653A CN110551653A CN201910832526.9A CN201910832526A CN110551653A CN 110551653 A CN110551653 A CN 110551653A CN 201910832526 A CN201910832526 A CN 201910832526A CN 110551653 A CN110551653 A CN 110551653A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Abstract
The invention provides a method for preparing mycoplasma bovis antigen without serum, which comprises the following steps: (1) incubating fresh SPF hatching eggs, culturing for 7-8 days, and selecting healthy chick embryos; (2) preparing mycoplasma bovis inoculation liquid; (3) inoculating selected healthy SPF (specific pathogen free) chick embryos of 7-8 days old to mycoplasma bovis inoculation liquid, placing the chick embryos in an incubator for continuous culture after inoculation, observing the state of the chick embryos, collecting inoculated dead chick embryos, and placing the chick embryos for 12-24 hours at the temperature of 2-8 ℃; (4) aseptically collecting the allantoic fluid of the dead chick embryo to obtain the mycoplasma bovis serum-free antigen. The invention can realize the serum-free production of the mycoplasma bovis antigen, avoid a plurality of problems caused by the use of animal serum, and has high titer of live bacteria for culturing the antigen.
Description
Technical Field
The invention relates to a method for preparing mycoplasma bovis antigen without serum.
Background
Bovine mycoplasmosis is an animal epidemic disease that seriously harms the cattle industry worldwide. The disease is caused by Mycoplasma bovis (m.bovis), and is clinically mainly manifested by calf pneumonia, arthritis, mastitis in dairy cattle and the like. The disease is currently prevalent worldwide, causing significant economic losses. Antibiotic therapy and vaccine immunization are important means for preventing and controlling bovine mycoplasmosis. In recent years, the emergence of more and more drug-resistant strains has brought about a serious challenge to the prevention and control of bovine mycoplasmosis.
Antigen production is a key link in vaccine preparation and affecting vaccine quality. At present, mycoplasma bovis antigen production culture mainly uses mycoplasma bovis liquid culture media, such as a modified KM2 culture medium, a beef heart soup culture medium, a modified Thiauucort's culture medium and the like. In the prior art, mycoplasma bovis culture depends on animal serum, and the culture medium contains a certain proportion of animal serum such as horse serum or pig serum, and the serum content is generally between 5% and 20%. The complex serum components and the difference among batches affect the quality control of the vaccine, the serum is easily polluted by microorganisms such as mycoplasma, the complex serum components are removed by a purification process in the later period, the side reaction of the serum-containing antigen to the cattle is easily increased, the immune effect is affected, and in addition, the use of the serum increases the vaccine production cost. The use of serum in the production of mycoplasma bovis vaccines presents a number of problems, and the preparation of mycoplasma bovis antigens with low or no serum is an important direction of development.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art that animal serum is used for preparing mycoplasma bovis antigens, and provides a serum-free preparation method of mycoplasma bovis antigens, which can overcome the defects of the prior art. The method of the invention uses the chick embryo to culture and produce the mycoplasma bovis antigen, realizes the serum-free production of the mycoplasma bovis antigen, can overcome a plurality of problems caused by the use of serum, has high culture titer, and reduces the production cost.
The invention provides a method for preparing mycoplasma bovis antigen without serum, which comprises the following steps:
(1) Incubating fresh SPF hatching eggs, culturing for 7-8 days, and selecting healthy chick embryos which are good in development, clear in blood vessels and vigorous in activity;
(2) Preparing mycoplasma bovis inoculation liquid;
(3) Inoculating selected healthy SPF (specific pathogen free) chick embryos of 7-8 days old to mycoplasma bovis inoculation liquid, placing the chick embryos in an incubator for continuous culture after inoculation, observing the state of the chick embryos, collecting inoculated dead chick embryos, and placing the chick embryos for 12-24 hours at the temperature of 2-8 ℃;
(4) Aseptically collecting the allantoic fluid of the dead chick embryo to obtain the mycoplasma bovis serum-free antigen.
Preferably, the steps (1) and (2) are intermodulation sequentially.
Preferably, in the step (2), the specific method for preparing mycoplasma bovis inoculation liquid comprises the following steps: and (3) recovering and subculturing the mycoplasma bovis strain, culturing for 18-36h in a constant-temperature incubator at 37 ℃, centrifuging when the color of the culture medium turns yellow or the pH value is reduced from 7.4 to 6.8-7.0, and resuspending the strain by using normal saline to obtain the mycoplasma bovis inoculation liquid.
Preferably, in the step (3), the inoculation is yolk sac inoculation, and the inoculation amount is 0.2ml for each chick embryo.
Preferably, in the step (3), the temperature of the incubator is 37.0-37.5 ℃, and the humidity is 50%.
Preferably, the method further comprises, after the step (4), the step of inoculating the collected allantoic fluid of chick embryos into healthy SPF chick embryos aged 7 to 8 days, inoculating and passaging for 3 to 5 times by the same method, and aseptically collecting the allantoic fluid of chick embryos.
The invention also provides application of the serum-free preparation method of the mycoplasma bovis antigen in mycoplasma bovis culture.
The invention also provides application of the serum-free preparation method of the mycoplasma bovis antigen in preparation of mycoplasma bovis vaccine antigens.
Compared with the prior art, the invention has the beneficial effects that: the produced mycoplasma bovis antigen does not contain animal serum components, and can overcome the problems of influence on vaccine quality control, microbial pollution, complicated purification process in later period, influence on immune effect caused by side reaction on a cow body and the like caused by using animal serum in the prior art. Compared with the complex preparation of the in vitro mycoplasma liquid culture medium, the SPF chick embryo has stable source, convenient acquisition and simple and convenient operation, and can obtain the high-titer serum-free mycoplasma bovis antigen.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the colony morphology of Mycoplasma bovis in allantoic fluid of chick embryos.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
The method for preparing the mycoplasma bovis antigen in a serum-free manner comprises the following steps:
(1) Incubating fresh SPF hatching eggs in an incubator (the temperature is 37.0-37.5 ℃ and the humidity is 50%), culturing for 7-8 days, lighting the eggs before inoculation, selecting healthy chick embryos which are well developed, clear in blood vessels and vigorous, removing the eggs without sperm, dead embryos and weak embryos, drawing the air chamber position of the healthy chick embryos in a dark room, and keeping the chick embryos for later use.
(2) Preparing mycoplasma bovis inoculum, namely resuscitating and passaging mycoplasma bovis strains, culturing for 18-36h in a constant-temperature incubator at 37 ℃ in an improved Thiaucourt's culture medium, centrifuging fresh mycoplasma bovis culture solution when the color of the culture medium turns yellow (or the pH value is reduced from 7.4 to 6.8-7.0), centrifuging at 8000rpm for 15min, re-suspending the bacteria with equal volume of normal saline to obtain an inoculation stock solution, and preparing 10 -1 normal saline diluent.
(3) Inoculating selected healthy SPF (specific pathogen free) chick embryos of 7-8 days old with mycoplasma bovis inoculation liquid, inoculating yolk sac with each chick embryo of 0.2ml, and inoculating 3 chick embryos at each dilution. After inoculation, the eggs are placed in an incubator to be continuously cultured for 12 days, the temperature of the incubator is 37.0-37.5 ℃, the humidity is 50%, the state of the chick embryos is observed every day, the inoculated dead chick embryos are collected, and the chick embryos are placed for 12-24 hours for standby at the temperature of 2-8 ℃.
(4) aseptically collecting the dead chick embryo allantoic fluid, namely the mycoplasma bovis serum-free antigen.
And (3) inoculating the stock solution of the chick embryo allantoic fluid obtained in the step (4) and 10 -1 physiological saline diluent into healthy SPF chick embryos of 7-8 days old, continuously inoculating and passaging for 3-5 times in the same step (3), and obtaining dead chick embryo allantoic fluid to perform viable bacteria titer determination.
Example 1
A method for preparing mycoplasma bovis antigen without serum comprises the following steps:
(1) incubating fresh SPF hatching eggs in an incubator (the temperature is 37.0-37.5 ℃ and the humidity is 50%), culturing for 7 days, lighting the eggs before inoculation, selecting healthy chick embryos which are well developed, clear in blood vessels and vigorous, discarding the eggs without sperm, dead embryos and weak embryos, drawing the air chamber position of the healthy chick embryos in a dark room, and keeping the chick embryos for later use.
(2) Preparing mycoplasma bovis inoculation liquid, namely recovering and passaging mycoplasma bovis PG45 strain (American culture collection ATCC, number ATCC 25523), culturing for 24 hours in a constant-temperature incubator at 37 ℃, wherein the culture medium is an improved Thiaumurt's culture medium, when the color of the culture medium turns yellow (or the pH value is reduced from 7.4 to 7.0), centrifuging fresh mycoplasma bovis culture liquid, centrifuging at 8000rpm for 15 minutes, re-suspending the bacteria with physiological saline with the same volume to obtain mycoplasma bovis inoculation stock solution, and preparing 10 -1 physiological saline diluent.
(3) The selected healthy 7-day-old SPF chick embryos are inoculated with the mycoplasma bovis inoculation liquid, yolk sacs are inoculated, 0.2 ml/piece is inoculated, and 3 chick embryos are inoculated in each dilution. After inoculation, the eggs are placed in an incubator to be continuously cultured for 12 days, the temperature of the incubator is 37.0-37.5 ℃, the humidity is 50%, the state of the chick embryos is observed every day, the inoculated dead chick embryos are collected and placed for 20 hours at 4 ℃.
(4) allantoic fluid from dead chick embryos was collected aseptically.
(5) Inoculating the obtained chick embryo allantoic fluid stock solution and 10 -1 physiological saline diluent into healthy SPF chick embryos of 7-8 days old, continuously inoculating and passaging for 3 times in the same step (3), harvesting death chick embryo allantoic fluid, and measuring viable cell titer.
Measuring average viable bacteria titer (CCU), namely taking 13 EP tubes, adding 0.9ml of culture medium into each tube, adding 0.1ml of to-be-measured substance into the 1 st tube, fully oscillating and uniformly mixing, then changing a gun head, sucking 0.1ml from the 1 st tube, adding into the 2 nd tube, sequentially diluting by 10 times, obtaining the dilution of the to-be-measured substance to be 10 -1 -10 -12 respectively, additionally arranging 1 culture medium control tube, carrying out standing culture in a 37 ℃ incubator for 3 times, observing color change every day, continuously observing for 10 days, and obtaining the highest dilution with color change, namely the viable bacteria titer of the culture, which is expressed by a Color Change Unit (CCU).
The colony morphology of Mycoplasma bovis in allantoic fluid of chick embryos was collected as shown in FIG. 1. The allantoic fluid collected was subjected to the measurement of viable cell titer of mycoplasma bovis antigen, and the results are shown in Table 1.
FIG. 1 shows the colony morphology of Mycoplasma bovis in allantoic fluid of chick embryos.
TABLE 1
The test result shows that the harvested chick embryo allantoic fluid contains mycoplasma bovis antigen, the colony morphology is typical 'fried egg-like', and accords with the characteristics of mycoplasma bovis colony morphology, the viable bacteria titer of the mycoplasma bovis antigen in the harvested chick embryo allantoic fluid reaches 10 8 -10 9 CCU/ml, and the average viable bacteria titer is 7.0 x 10 8 CCU/ml.
Comparative test example
The method of the invention and the conventional serum-containing mycoplasma bovis culture medium (10% serum bovine heart broth culture medium, 20% serum modified KM2 culture medium) are respectively applied to culture mycoplasma bovis PG45 strain, and the average viable bacteria titer of the obtained mycoplasma bovis antigen is compared (10-fold gradient dilution determination of the culture, 1.0ml dilution system or 5.0ml dilution system). The results of the comparison are shown in Table 2.
The formula of the 10% serum beef heart soup culture medium is as follows: 562.5ml of 1% hydrolyzed milk protein Hank's solution, 337.5ml of beef heart soup, 20ml of 25% fresh yeast extract, 1ml of penicillin (20 ten thousand IU/ml), 2.5ml of 1% phenol red, 1ml of 10% thallium acetate and 100ml of sterile horse serum, and after mixing, the pH value is adjusted to 7.4 by using sterilized 1M NaOH.
The formula of the 20% serum improved KM2 culture medium is as follows: MEM 5g, glucose 0.4g, sodium pyruvate 0.2g, hydrolyzed milk protein 5.1g, 1% phenol red 2.5ml, 25% fresh yeast extract 20ml, penicillin (20 ten thousand IU/ml)1ml, 10% thallium acetate 1ml, sterile horse serum 200ml, mixing, adding deionized water to 1000ml, and adjusting pH to 7.4 with sterilized 1M NaOH.
TABLE 2
| culture method/culture medium | Serum content | Viable bacteria titer CCU/ml |
| example 1 | 0％ | 7.0×108 |
| Beef heart soup culture medium | 10％ | 1.0×108 |
| Improved KM2 culture medium | 20％ | 1.0×107 |
As can be seen from Table 2, the viable titer of Mycoplasma bovis obtained by the method of the invention is obviously higher than that of the conventional serum-containing Mycoplasma bovis culture medium (10% serum Cor bovis Seu Bubali soup culture medium, 20% serum modified KM2 culture medium). The result shows that compared with the conventional serum-containing mycoplasma bovis culture medium, the method has the advantages of no serum and high viable bacteria titer when the mycoplasma bovis antigen is cultured.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for preparing mycoplasma bovis antigen without serum is characterized in that: the method comprises the following steps:
(1) Incubating fresh SPF hatching eggs, culturing for 7-8 days, and selecting healthy chick embryos which are good in development, clear in blood vessels and vigorous in activity;
(2) Preparing mycoplasma bovis inoculation liquid;
(3) Inoculating selected healthy SPF (specific pathogen free) chick embryos of 7-8 days old to mycoplasma bovis inoculation liquid, placing the chick embryos in an incubator for continuous culture after inoculation, observing the state of the chick embryos, collecting inoculated dead chick embryos, and placing the chick embryos for 12-24 hours at the temperature of 2-8 ℃;
(4) aseptically collecting the allantoic fluid of the dead chick embryo to obtain the mycoplasma bovis serum-free antigen.
2. The method of claim 1, wherein: and (3) sequentially intermodulation of the step (1) and the step (2).
3. The method of claim 1, wherein: in the step (2), the specific method for preparing mycoplasma bovis inoculation liquid comprises the following steps: and (3) recovering and subculturing the mycoplasma bovis strain, culturing for 18-36h in a constant-temperature incubator at 37 ℃, centrifuging when the color of the culture medium turns yellow or the pH value is reduced from 7.4 to 6.8-7.0, and resuspending the strain by using normal saline to obtain the mycoplasma bovis inoculation liquid.
4. The method of claim 1, wherein: in the step (3), the inoculation is yolk sac inoculation, and the inoculation amount is 0.2ml for each chick embryo.
5. the method of claim 1, wherein: in the step (3), the temperature of the incubator is 37.0-37.5 ℃, and the humidity is 50%.
6. The method of claim 1, wherein: and (4) inoculating the collected chick embryo allantoic fluid into healthy SPF chick embryos of 7-8 days old, inoculating and passaging for 3-5 times by using the same method, and aseptically collecting the chick embryo allantoic fluid.
7. Use of a serum-free preparation method of mycoplasma bovis antigens according to any one of claims 1-7 in mycoplasma bovis culture.
8. Use of a serum-free preparation method of mycoplasma bovis antigens according to any one of claims 1-7 in the preparation of mycoplasma bovis vaccine antigens.
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